Use of NF-κB inhibition in combination therapy for cancer

ABSTRACT

The use of NF-κB inhibitors to enhance the cytotoxic effects of chemotherapy or radiation therapy in the treatment of neoplastic conditions is described.

This application is a continuation of U.S. patent application Ser. No.10/979,403 filed Nov. 2, 2004, which in turn is a continuation of U.S.patent application Ser. No. 08/959,160, filed Oct. 28, 1997, now issuedas U.S. Pat. No. 6,831,057, the disclosures of both of which areincorporated by reference herein in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant NumbersAI35098 and 1F32-CA69790-01 from the National Institutes of Health andGrant Number DAMD17-94-J-4053 from the Department of the Army. The USGovernment has certain rights to this invention.

BACKGROUND OF THE INVENTION

The inducible transcription factor Nuclear Factor-kappa B (NF-κB)participates in the regulation of multiple cellular genes, includingmany involved in the immune and inflammatory processes (for example,GM-CSF, IL-6, IL-8 and IL-2). NF-κB is a member of the rel family ofprotein complexes, and is activated by cellular exposure to variousfactors, including phorbol 12-myristate 13-acetate (PMA),lipopolysaccharide (LPS), interleukin-1 (IL-1), tumor necrosis alpha(TNFα), and ultraviolet radiation. See Baldwin, Annu. Rev. Immunol.14:649 (1996). NF-κB has also been implicated in the transcriptionalactivation of several viruses, including HIV-1 (Nabel et al., Nature326:711 (1987); Kaufman et al., Mol. Cell. Biol., 7:3759 (1987)). Thevarious signaling pathways that can control the activation of NF-κB arenot all clearly understood. It is apparent that different inducers caninitiate their pathways through distinct receptors.

The latent cytoplasmic form of NF-κB is associated with a cytoplasmicinhibitory protein called IκB. Baeuerle and Baltimore, Science 242:540(1988); PCT US92/04073 (WO 92/20795). The release of NF-κB from IκBresults in the rapid appearance of the active form of NF-κB in the cellnucleus. Genes regulated by NF-κB can be transcriptionally activatedminutes after exposure of the cell to the appropriate inducer.Activation of NF-κB after exposure of a cell to an inducer is correlatedwith the hyperphosphorylation of IκBα and its subsequent degradation. AsIκB is diminished in the cytoplasm, NF-κB increases in the nucleus.Phosphorylation of IκB was once thought to lead to dissociation fromNF-κB and subsequent proteolysis of IκB. Beg and Baldwin, Genes Dev.7:2064 (1993). More recently, it has been proposed that the proteasomeis responsible for signal-mediated degradation of IκBα and IκBβ.Baldwin, Annu. Rev. Immunol. 14:649 (1996). Phosphorylation of IκBapparently renders the molecule susceptible to proteolysis.

Activation of NF-κB has been suggested as playing a pathological role inthe development of autoimmune diseases and chronic inflammatory diseasessuch as rheumatoid arthritis, and in acute situations such as septicshock.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of enhancing thecytotoxic effects of an antineoplastic chemotherapeutic agent. Atherapeutically effective amount of NF-κB inhibitor is administered inconjunction with the chemotherapeutic agent, so that the cytotoxiceffect of the chemotherapeutic agent is increased compared to that whichoccurs in the absence of NF-κB inhibitor.

A further aspect of the present invention is a method of enhancingchemotherapeutic cytotoxicity in a subject treated with achemotherapeutic agent. A therapeutically effective amount of NF-κBinhibitor is administered to a subject in conjunction with achemotherapeutic agent. The cytotoxic effect of the chemotherapeuticagent is increased compared to that which occurs in the absence of NF-κBinhibitor.

A further aspect of the present invention is a method of enhancing thecytotoxic effect of TNFα, by administering a therapeutically effectiveamount of NF-κB inhibitor in conjunction with TNFα. The cytotoxic effectof TNFα is increased compared to that which occurs in the absence ofNF-κB inhibitor.

A further aspect of the present invention is a method of enhancingchemotherapeutic cytotoxicity in a subject treated with TNFα, byadministering a therapeutically effective amount of NF-κB inhibitor inconjunction with TNFα. The cytotoxic effect of TNFα is increasedcompared to that which occurs in the absence of NF-κB inhibitor.

A further aspect of the present invention is a method of screening acompound for the ability to reduce the anti-apoptotic protective effectsof an NF-κB induced anti-apoptotic gene. A population of test cells isexposed to an anticancer treatment and a test compound, and the cellviability following the exposure is assessed. Reduced cell viability,compared to that which occurs in cells treated only with the anticancertreatment, indicates that the test compound reduces the anti-apoptoticeffects of an NF-κB induced anti-apoptotic gene.

The foregoing and other objects and aspects of the present invention areexplained in detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A graphs the percentage cell survival over time in fibrosarcomacells expressing the super-repressor IκBα (HT1080I; solid diamonds) orin control fibrosarcoma cells containing the empty expression vector(HT1080V; open squares), after exposure to TNFα. (Data shown are themean of three independent experiments ±SD and the percentage cellsurvival was defined as the relative number of THF treated versusuntreated cells). TNF-mediated apoptosis was increased over time incells expressing the super-repressor IκBα, compared to control cells.

FIG. 1B portrays the detection of TNF-induced apoptosis by TUNELstaining in HT1080V (control; V) or HT1080I (super-repressor IκBαexpressing; I) cells that were either untreated (−TNF) or stimulated(+TNF) with TNFα. Positive cells show the condensed morphology typicalof apoptotic cells.

FIG. 2A graphs cell survival over time in HT1080V (control) or HT1080I(super-repressor IκBα expressing) cells pre-treated with IL-1 prior toexposure to TNFα and CHX. Interleukin-1 (IL-1) pre-treatment inhibitedTNFα+CHX-induced apoptosis in HT1080V cells. Open squares representHT1080V cells without IL-1 pretreatment; closed squares representHT1080V cells pretreated with IL-1; circles represent HT1080I cellswithout IL-1 pretreatment; triangles represent HT1080I cells pretreatedwith IL-1.

FIG. 2B compares the extent of TNFα-induced apoptosis in HT1080I cells(expressing the super-repressor IκBα) co-transfected to express LacZ,p65 and p50 (hatched bars) or co-transfected with the LacZ and emptyvectors. After 40 hours, cells were treated with differentconcentrations of TNFα for an additional 24 hours. (Results are from themean±SD of two experiments). Expression of the p50 and RelA/p65 NH-κBsubunits restored cell resistance to TNFα-induced apoptosis in cellsexpressing the super-repressor IκBα.

FIG. 3A provides the results of electrophoretic mobility-shift assays(EMSA) that indicate NF-κB nuclear translocation. HT1080V (control; V)or HT1080I (expressing the super-repressor IκBα; I) cells were treatedwith daunorubicin, staurosporine, or irradiation. (Lanes 1-5,daunorubicin; Lanes 6-9, staurosporine; and Lanes 10-13, ionizingradiation). In HT1080V control cells, both daunorubicin (lanes 1-3) andionzing radiation (lanes 10-11) activated NF-κB and resulted in nucleartranslocation over time. In contrast, activation of NF-κB bydaunorubicin and ionizing radiation was blocked in HT1080I cells (lanes4-5 and 12-13, respectively). Treatment with staurosporine did notresult in NF-κB nuclear translocation in HT1080V control cells (lanes6-9).

FIG. 3B compares cell survival of HT1080V cells (control; solid bars)and HT1080I cells (expressing the super-repressor IκBα; hatched bars)treated with varying concentrations of daunorubicin for 24 hours.(Results are from the mean of four separate experiments). Theoverexpression of super-repressor IκBα enhanced cell killing bydaunorubicin.

FIG. 3C compares cell survival of HT1080V cells (control; solid bars)and HT1080I cells (expressing the super-repressor IκBα; hatched bars) 14days after treatment with ionizing radiation for the indicated doses(Gy). (Results shown represent three independent experiments and areexpressed as the mean±SD). The overexpression of super-repressor IκBαenhanced cell killing by ionizing radiation.

FIG. 4 graphs tumor growth in mice after treatment with theantineoplastic CPT-11 (irinotecan), with and without the adenoviraldelivery of the super-repressor IκB. Tumors receiving both IκB andCPT-11 showed essentially no growth during the 20 days after treatment.

DETAILED DESCRIPTION

Many cells are resistant to stimuli which are otherwise capable ofinducing apoptosis, but the mechanisms involved are not fullyunderstood. It is demonstrated herein that tumor necrosis factor (TNF),ionizing radiation, and anti-cancer chemotherapeutic compounds (such asdaunorubicin), activate NF-κB and that this activation of NF-κB protectsthe cell against the cytotoxic effects of these treatments. Byinhibiting NF-κB nuclear translocation, the present inventors enhancedthe apoptotic killing achieved by these reagents; no such enhancement ofapoptosis by NF-κB inhibitors was seen with apoptotic stimuli that donot activate NF-κB. These results indicate that NF-κB activationprovides cellular resistance to killing by some apoptotic reagents, andprovide a method to improve the efficacy of various cancer therapies.

Observations that NF-κB is activated by certain apoptotic stimulisuggests that this transcription factor plays a role in mediatingaspects of programmed cell death. Baldwin, Annu. Rev. Immunol. 14:649(1996); Verma et al., Genes Dev. 9:2723 (1995); Siebenlist et al., Annu.Rev. Cell Biol. 10:405 (1994); Liou and Baltimore, Curr. Opin. CellBiol. 5:477 (1993); Baeuerle and Henkel, Annu. Rev. Immunol. 12:141(1994). PCT patent application WO 97/30083 describes anti-apoptoticproteins capable of inhibiting NF-κB, and describes methods ofdecreasing apoptosis by administering such anti-apoptotic proteins. Ananti-apoptotic function of NF-κB is also suggested by the finding thatmice lacking the NF-κB p65/RelA gene die embryonically from extensiveapoptosis within the liver. Beg et al., Nature 376:167 (1995).Furthermore, it has been observed that many cells which respond to TNF(a strong activator of NF-κB) are resistant to cell killing, and thatTNF killing is enhanced in the presence of protein synthesis inhibitors.Holtmann et al., Immunobiology 177:7 (1988).

The present inventors investigated whether activation of NF-κB isprotective against the apoptotic killing induced by TNF in a model cellsystem. Their studies used the human fibrosarcoma cell line HT1080(which is relatively resistant to killing by TNF; Wang and Baldwin,unpublished data). In order to potentially block the activation of NF-κBin response to TNF stimulation, an HT1080 cell line (HT1080I) expressinga super-repressor form of the NF-κB inhibitor IκBα was established. Thesuper-repressor IκBα contains serine to alanine mutations at residues 32and 36, which inhibit signal-induced phosphorylation and which inhibitsubsequent proteasome-mediated degradation of IκBα. This mutant IκBαprotein acts as a super-repressor because it binds to NF-κB and inhibitsDNA binding as well as nuclear translocation, but is unable to respondto cellular signals such as those induced by TNF. See Brockman et al.,Mol. Cell. Biol. 15:2809 (1995); Brown et al., Science 267:1485 (1995);Traenckner et al., EMBO J. 14:2876 (1995); DiDonato et al., Mol. Cell.Biol. 16:1295 (1996); Palombello et al., Cell 78:773 (1994); Chen etal., Genes Dev. 9:1586(1995). A control line (HT1080V) was establishedthat contained the empty vector and the hygromycin selectable marker.TNFα-induced NF-κB activation, as measured by DNA binding of nuclearextracts, was effectively blocked by the super-repressor IκBα in HT1080Icells as compared to the control cell line (see FIG. 3A).

As shown in FIG. 1A, TNFα is much more effective at inducing apoptosisin the IκBα super-repressor expressing cells (HT1080I) than the controlcell line (HT1080V). Similar results were obtained using pooled clonesof HT1080V or HT1080I cells (data not shown), indicating that theresults were due to overexpression of the super-repressor IκBα, and notto clonal variation. That cells were killed through apoptosis wasconfirmed by using the deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) assay which measures DNA strand breaks and istherefore diagnostic for cells undergoing apoptosis. Apoptosis wasobserved only in the HT1080I cells treated with TNF (FIG. 1B). Thus,expression of a super-repressor form of IκBα, known to be highlyeffective at inhibiting NF-κB nuclear translocation, was found toenhance the ability of TNF to initiate apoptosis in cells that arenormally resistant to this cytokine. These data indicate, therefore,that the activation of NF-κB by TNF is protective, providing protectionagainst the cell killing induced by this apoptotic agent.

To exclude the possibility that the expression of the super-repressorform of IκBα leads to a function that is different from the inhibitionof NF-κB, the present inventors confirmed the requirement for NF-κB inthe inhibition of TNF-induced apoptosis. The pre-treatment of HT1080Vcells (control cells) with interleukin-1 (IL-1, an activator of NF-κBthat does not initiate apoptosis) blocked the subsequent killing ofthese cells induced by combined cycloheximide (CHX) and TNF treatment.As shown in FIG. 2A, IL-1 pre-treatment of the HT1080V cells protectedagainst CHX+TNF-induced killing, compared to cells which were notpretreated. As a control and to show that it was NF-κB that wasresponsible for the protection against apoptosis, it was demonstratedthat IL-1 had no protective effect on the HT1080I cell line, in whichNF-κB activation is blocked by the expression of the super-repressor IκBα (FIG. 2A). Holtmann et al. reported that IL-1 pretreatment was capableof blocking TNF-mediated killing (Immunobiology 177:7 (1988)). Thepresent inventors' results indicate that the mechanism behind theability of IL-1 to inhibit TNT-responsive apoptosis is the induction ofNF-κB.

The present inventors additionally determined that proteasome inhibitorsenhanced cell killing of HT1080 cells in response to TNF treatment. Thedegradation of IκB that results in the release of active NF-κB iscontrolled by the proteasome following inducible phosphorylation andsubsequent ubiquitination of IκB (Palombello et al., Cell 78:773 (1994);Chen et al., Genes Dev. 9:1586(1995)). Proteasome inhibitors of thepeptide aldehyde category are potent inhibitors of NF-κB activation(Palombello et al., Cell 78:773 (1994); Chen et al., Genes Dev.9:1586(1995)). The present inventors demonstrated that, in adose-dependent fashion, the proteasome inhibitor MG132 (Z-leu-leu-leu-H)strongly enhanced the killing of HT1080V cells in response to TNF (datanot shown).

If NF-κB inhibition sensitizes cells to TNF killing, then overexpressionof NF-κB subunits should restore protection against cell killing in thepresent HT1080I model. Vectors encoding the p50 and RelA/p65 subunits ofNF-κB or the empty CMV vector control were transfected into the HT1080Icells, and the cells were stimulated with TNF. As expected, the vectoralone did not provide protection against cell killing induced by TNF(FIG. 2B). However, expression of the NF-κB p50 and RelA/p65 subunitsprovided protection against TNF-induced apoptosis, indicating that it isNF-κB that is blocked by the super-repressor IκBα, and that NF-κBexpression blocks programmed cell death. Additional evidence that NF-κBis required for protection against TNF-induced apoptosis is shown by thefact that embryonic fibroblasts from RelA/p65 null mice (Beg et al.,Nature 376:167 (1995)) are killed by TNF with a much higher frequencythan those from wild-type animals (data not shown; see also Beg andBaltimore, Science 274:782 (1996)).

Slater et al. reported that in thymocytes (which constitutively expressNF-κB) the intensity of the NF-κB/rel protein-DNA complex was enhancedafter exposure to etoposide (an agent known to induce apoptosis inthymocytes). Slater et al., Biochem J. 312:844 (1995). These authorsconcluded that NF-κB expression is correlated with subsequent apoptosis.Bessho et al. examined the effect of pyrrolidine dithiocarbamate (PDTC)on the induction of apoptosis by a variety of agents. Bessho et al.,Biochem, Pharm. 48:1883 (1994). They reported that treatment of a humanpromyelocytic leukemia cell line with etoposide or1-beta-D-arabinofaranosylcytosine induced NF-κB activation, withapoptosis occurring in 3-4 hours. Addition of PDTC was reported to blockNF-κB activation; these authors concluded that etoposide- anddexamethasone-induced apoptosis in human thymocytes was also blocked byPDTC, and that the activation of NF-κB plays a role in the apoptoticprocess of human hematopoietic cells.

Many cancer therapies function to kill neoplastic cells throughapoptotic mechanisms; cellular resistance to apoptosis providesprotection against cytotoxic therapies (reviewed in Fisher, Cell, 78:539(1994)). To determine if other apoptotic stimuli in addition to TNFactivate NF-κB and induce a protective effect of NF-κB, the presentinventors analyzed ionizing radiation-, daunorubicin- andstaurosporine-treated cells. Ionizing radiation has been shown toactivate NF-κB in several cell types (Singh and Lavin, Mol. Cell. Biol.10:5279 (1990); Brach et al., J. Clin. Invest. 88:691 (1991)). Thepresent inventors first investigated whether ionizing radiation, thechemotherapeutic compound daunorubicin, or staurosporine activated NF-κBin the HT1080V cells and in the HT1080I cells. Data shown in FIG. 3Ashows that both daunorubicin (lanes 1-3) and ionizing radiation (lanes10-11) activated NF-κB. Importantly, the ability of these two stimuli toactivate NF-κB in HT1080I cells (expressing the super-repressor IκBα)was blocked (FIG. 3A, lines 4-5 and lane 13). In contrast, staurosporine(a cytotoxic agent that is not used in cancer treatment) did notactivate NF-κB (FIG. 3A, lanes 6-9).

The present inventors also tested whether ionizing radiation-,daunorubicin-, and staurosporine-induced cell killing could be enhancedby the inhibition of NF-κB activity. Daunorubicin (FIG. 3B) and ionizingradiation (FIG. 3C) were found to induce NF-κB activation; thecytotoxicity of these agents was enhanced in HT1080I cells (expressingthe super-repressor IκBα) compared to control cells. However, apoptosisinduced by staurosporine was not enhanced by the expression of IκBα.(data not shown), consistent with the present inventors' observationthat staurosporine does not effectively activate NF-κB (FIG. 3A). Thus,the activation of NF-κB is part of the cellular response to a variety ofcytotoxic agents, and provides protection against the apoptosis causedby such agents. The present inventors data additionally indicate thatcell killing by vincristine is augmented by the inhibition of NF-κB(data not shown). Tumor growth after treatment with the antineoplasticCPT-11 (irinotecan), with and without the adenoviral delivery of thesuper-repressor IκB, was also studied. CPT-11 activated NF-κB (data notshown). Tumors receiving both IκB and CPT-11 showed essentially nogrowth during the 20 days after treatment (FIG. 4); cytotoxicity of thechemotherapeutic agent was increased when combined with thesuper-repressor IκB. Cell-killing was by apoptosis (data not shown).

The present data indicate that the activation of NF-κB by TNF, ionizingradiation, and chemotherapeutic agents (such as daunorubicin,vincristine and irinotecan) protects cells against the apoptotic cellkilling induced by these stimuli. Distinct signaling pathways initiatedby TNF engagement of its receptor lead to activation of both apoptosisand NF-κB, and NF-κB does not play a positive role in the induction ofapoptosis (Tartaglia and Goeddel, Immunol. Today 13:151 (1992);Vandenabeele et al., Trends Cell. Biol. 5:392 (1995); Hsu et al., Cell81:495 (1995); Hsu et al., Cell 84:299 (1996); Chinnaiyan et al., Cell81:505 (1995); Goncharov et al., Cell 84:803 (1996); Hsu et al.,Immunity 4:387 (1996)). In the case of ionizing radiation anddaunorubicin the activation of apoptosis appears to be initiated byceramide production (Bose et al., Cell 82:405 (1992); Jaffezou et al.,EMBO J. 15:2417 (1996); Haimovitz-Freedman et al., J. Exp. Med. 180:525(1994); Santana et al., Cell 86:189 (1996)), and the cytotoxic effectsof TNF have been reported to require ceramide activation (Kolesnick andGolde, Cell 77:325 (1994)). Ceramide alone has been shown to lead toapoptosis (Obeid et al., Science 259:1769 (1993)) but the details ofthis apoptotic pathway are not fully understood. It should be noted thatseveral groups have suggested that NF-κB may function pro-apoptoticallyunder some conditions and in certain cell lines (Jung et al Science268:1619 (1995); Lin et al., J. Cell Biol. 131:1149 (1995); Grimm etal., J. Cell Biol. 134:1 (1996)).

Growing evidence indicates that a variety of anticancer agents killthrough programmed cell death. Resistance to anticancer therapiesappears to be mediated by resistance to apoptosis (Fisher, Cell 78:539(1994)). The present data demonstrate that several anticancer agents maybe less effective at inducing programmed cell death because of theirconcomitant activation of NF-κB.

As used herein, “NF-κB activation” refers to NF-κB-mediatedup-regulation of genes which are directly or indirectly under thecontrol of an NF-κB binding site. In functional terms, NF-κB activationcomprises the binding of NF-κB to κB regulatory sequences in the DNA ofa cell, so that transcription of the operatively associated gene isinduced (other factors acting in combination with NF-κB may be requiredto initiate transcription).

“NF-κB inhibition” refers to the prevention of NF-κB binding to NF-κBbinding sites in nuclear DNA. NF-κB inhibition does not necessarilyimply that all NF-κB-induced transcription is prevented, but rather thatsuch transcription occurs at levels substantially less than that whichwould occur in the absence of NF-κB inhibition. For example, NF-κBinhibition may result in a 30% reduction in NF-κB-induced transcriptionof a gene, more preferably a 40%, 50%, or 60% reduction in NF-κB-inducedtranscription of a gene, and even more preferably a 70%, 80%, 90%, 95%or greater reduction in NF-κB-induced transcription of a gene.

An “NF-κB inhibiting substance”, “NF-κB inhibitor”, or “NF-κB inductioninhibitor” refers to a substance capable of preventing or reducing NF-κBbinding to NF-κB binding sites in nuclear DNA, compared to that whichwould occur in the absence of the NF-κB inhibiting substance.

According to the present invention, methods or compounds that inhibitactivation or nuclear translocation of NF-κB, are utilized to enhancethe cellular apoptosis caused by various anti-cancer therapies. Methodsaccording to the present invention include treatment of a subject by theadministration of an NF-κB inhibitor in conjunction with an anti-cancertherapy, where the anti-cancer therapy alone would induce theanti-apoptotic effects of NF-κB. Addition of the NF-κB inhibitor permitsor enhances the apoptotic cell-killing effects of the anti-cancertherapy. Such combined therapy is useful in the treatment of tumors andother neoplastic growths and conditions. Thus, combined therapy thatinhibits NF-κB function in the presence of apoptotic stimuli lowers theapoptotic threshold of neoplastic cells and provides a more effectivetreatment against resistant forms of cancer. Additionally, theinhibition of NF-κB function in association with TNF treatment enhancesthe previously limited usefulness of this cytokine as an anti-tumoragent.

Methods of the present invention comprise the co-administration of ananti-cancer agent or treatment that induces NF-κB activation, and anNF-κB inhibitor. Such concurrent therapy enhances the cytotoxic abilityof the anti-cancer agent, compared to that which would occur in theabsence of NF-κB inhibitor. Any suitable NF-κB inhibitor or method ofinhibiting NF-κB activation may be used in the present methods. One suchmethod of inhibition is the delivery of the super-repressor IκBα tocells undergoing anti-cancer treatment, including via delivery ofnucleotide sequences encoding the super-repressor IκBα. A variety ofagents (examples discussed below) are known as NF-κB inhibitors,including proteasome inhibitors, inhibitors of ubiquitin conjugation,inhibitors of proteasome peptidases, and protease inhibitors.Additionally, the use of antisense oligonucleotides to control theexpression of cellular components is known in the art, and may beutilized in the present methods to reduce the expression of NFκB or itssubunits. (Antisense oligonucleotides that hybridize to NF-κB mRNA, andtheir therapeutic use to suppress processes that depend on activation ofNF-κB, are described in WO95/35032).

One proteolytic pathway in the cytosol involves the covalent conjugationof cellular proteins with the ubiquitin polypeptide (see, e.g., Hiershkoet al., Ann. Rev. Biochem. 61:761 (1992)). The conjugated proteins arethen hydrolyzed by a proteolytic complex containing a degradativeparticle called the proteasome (Goldberg et al, Nature, 357:375 (1992)).The proteasome is composed of multiple subunits and contains at leastthree different peptidases (Goldberg et al., Nature 357:375 (1992);Goldberg, Eur. J. Biochem. 203:9 (1992); Orlowski, Biochemistry 29:10289(1990); Rivett et al., Arch. Biochem. Biophys. 218:1 (1989); Tanaka etal., New Biol. 4:1 (1992)). These peptidases are referred to aschymotrypsin-like peptidase, the trypsin-like peptidase, and thepeptidylglutamyl peptidase.

WO 95/25533 describes a method of reducing the cellular content andactivity of NF-κB by contacting cells with inhibitors of proteasomefunction or ubiquitin conjugation; inhibition of proteolysis is achievedby interfering with the ubiquitin dependent pathway at any of severalpossible steps (e.g., interfering with the activity of the proteasomecomplex, or interfering with the activity of a proteasome component).Various inhibitors of ubiquitin conjugation to proteins are also known(Wilkinson et al., Biochemistry 29:7373 (1990)).

Various inhibitors of the peptidases of the proteasome have beenreported (Dick et al., Biochemistry 30:2725 (1991); Goldberg et al.,Nature 357:375 (1992); Goldberg, Eur. J. Biochem. 203:9 (1992);Orlowski, Biochemistry 29:10289 (1990); Rivett et al., Arch. Biochem.Biophys. 218:1 (1989); Rivett et al., J. Biol. Chem. 264:12215 (1989);Tanaka et al., New Biol. 4:1 (1992)). These inhibitors include knowninhibitors of chymotrypsin-like and trypsin-like proteases, andinhibitors of thiol (or cysteine) and serine proteases. Some endogenousinhibitors of proteasome activities have also been isolated (Murakami etal., Proc. Natl. Acad. Sci. USA 83:7588 (1986); Li et al., Biochemistry30:9709 (1991); Golberg, Eur. J. Biochem. 203:9 (1992); Aoyagi et al.,Proteases and Biological Control, Cold Spring Harbor Laboratory Press,pp. 429 (1975); Siman et al., WO 91/13904). Potential inhibitors of thechymotrypsin-like activity of the proteasome have been tested (Vinitskyet al., Biochemistry 31:9421 (1992); Orlowski et al. Biochemistry32:1563-1572 (1993); see also WO 95/25533).

Natural and chemical protease inhibitors include peptides containing anα-diketone or an α-keto ester, peptide chloromethyl ketones,isocoumarins, peptide sulfonyl fluorides, peptidyl boronates, peptideepoxides, and peptidyl diazomethanes (see e.g., Angelastro et al., J.Med. Chem. 33:11 (1990); Bey et al., EPO 363,284; Bey et al., EPO364,344; Grubb et al., WO 88/10266; Higuchi et al., EPO 393,457; Ewoldtet al., Molecular Immunology 29:713 (1992); Hernandez et al., J.Medicinal Chem. 35:1121 (1992); Vlasak et al, J. Virology 63:2056(1989); Hudig et al., J. Immunology 147:1360 (1991); Odakc et al,Biochemistry 30:2217 (1991); Vijayalakshmi et al., Biochemistry 30:2217(1991); Kam et al., Thrombosis and Haemostasis 64:133 (1990); Powers etal., J. Cell. Biochem. 39:33 (1989); Powers et al., ProteinaseInhibitors, Barrett et al. (Eds.), Elsevier, pp. 55-152 (1986); Powerset al., Biochemistry 29:3108 (1990); Oweida et al., Thrombosis Research58:391 (1990)).

Sherman et al. report that pyrrolidine dithiocarbamate (PDTC) is aninhibitor of NF-κB activation. Sherman et al., Biochem. Biophys. Res.Comm. 191:1301 (1993). Anti-inflammatory steroids are suggested as NF-κBinduction inhibitors in PCT/US95/12534 (WO/96/10402), including but notlimited to predonsone, prednisolone, methyl prednisolone, dexamethasone,predisone, deoxycorticosterone, cortisone, hydrocortisone.Nonglucocorticoid lazaroids are also suggested for use as NF-κBinduction inhibitors. Novel amides that are inhibitors of NF-κB DNAbinding are described in WO 97/23457. Japanese patent applications JP7291859 and JP 7291860 describe chemical inhibitors of NF-κB activity.Additional compounds stated to be useful for inhibiting the action ofNF-κB in the nucleus are provided in WO 95/01348. Antisenseoligonucleotides that hybridize to NF-κB mRNA, and their therapeutic useto suppress processes which depend on activation of NF-κB, is describedin WO95/35032.

The administration of an NF-κB inhibitor to neoplastic cells accordingto the present invention may also be accomplished by gene therapy, e.g.,by transfecting a cell to be treated with a nucleic acid sequenceencoding an NF-κB inhibitor. The sequence may be incorporated, accordingto standard methods, into a mammalian vector suitable for transfectingmammalian cells in vivo, such as an adenovirus, a modified vacciniavirus, an Epstein Barr virus, or the like. The use of retroviral vectorsparticularly replication-defective retroviral vectors) for gene deliveryis well-known in the art and may be used to transform cells fortreatment according to the present invention. Alternative methods oftargeted gene delivery include DNA-protein conjugates, liposomes, andother methods as are known in the art.

Methods for introducing genes into mammalian cells are known in the art.In the present methods, the vector is introduced to the subject,preferably by direct administration (such as by injection) to the tumoror area to be treated. It is appreciated that the vector may beintroduced into nearby non-target cells as well as the target cells whenthe vector is injected into the treatment area.

Glucocorticoids are widely used as immune and inflammatory suppressants,and inhibit NF-κB (Ray and Prefontaine, Proc. Natl. Acad. Sci. USA91:752 (1994); Mukaida et al., J. Biol. Chem. 269:13289 (1994);Scheinrnan et al., Mol. Cell Biol. 15:943 (1995); Caldenhoven et al.,Mol. Endocrinol. 9:401 (1995); Scheinman et al., Science 270:283 (1995);Auphan et al, Science 270:286 (1995)). Glucocorticoids are presentlyused as part of a therapy for certain hematological malignancies (DeVitaet al., Cancer Res, 47:5810 (1987); Schartzman and Cidlowski,Endocrinol. Rev. 14:133 (1993)).

NF-κB inhibitors used according to the methods of the present inventionmay be administered by any suitable means, as would be apparent to oneskilled in the art, including systemically (e.g., intravenously) orlocally (e.g., injected into a tumor, a tumor cyst, tissues immediatelysurrounding a tumor, or into an anatomic compartment containing atumor). For example, where a therapeutically effective amount of NF-κBis utilized as an adjunct to chemotherapy, the NF-κB may be administeredlocally to a tumor or neoplasm (or the immediately surrounding tissue).Where a chemotherapeutic agent is delivered systemically, for example, atherapeutically effective amount of NF-κB inhibitor may be administeredsystemically by intravenous injection. Alternatively, the NF-κBinhibitor may be administered by a route different than that used forthe chemotherapeutic agent (e.g., local administration of NF-κBinhibitor to a tumor combined with systemic administration of achemotherapeutic agent).

The present inventors have determined that anti-cancer treatments canactivate NF-κB, and that this activation of NF-κB suppresses apoptosis.The co-administration of NF-κB inhibitors with an anti-cancer treatment,to enhance the apoptotic effects achieved by the cancer treatment, is anaspect of the present invention.

A further aspect of the present invention is a screening assay toidentity compounds that inhibit NF-κB induced anti-apoptotic genes(i.e., genes that are induced by NF-κB and that increase the resistanceof the cell to apoptotic stimuli, and more specifically, increase theresistance of the cell to the apoptotic effects of an anti-cancertreatment). Such a screening method may comprise exposing a populationof test cells to both an anti-cancer treatment and a test compound, anddetermining cell viability after such exposure. Reduced cell viabilityor reduced cell survival in the test cells (compared to that whichoccurs or would be expected to occur after treatment with theanti-cancer treatment alone) indicates that the test compound is capableof inhibiting the anti-apoptotic effects of a gene that is controlled byNF-κB. (The test compound may first be screened to document that it isnot an inhibitor of NF-κB activation; any observed reduction in cellsurvival would thus not be due to inhibition of NF-κB activation, but toeffects on the NF-κB induced gene itself.)

A further aspect of the present invention is a method of identifyingNF-κB induced genes, including those that have an anti-apoptotic effect.Such a method utilizes techniques known in the art for constructing acDNA library of a cell, such as polymerase chain reaction (PCR). (See,e.g., U.S. Pat. No. 5,589,622 to Gurr et al.) The cDNA library of a cellexposed to an anticancer therapy that is known to activate theanti-apoptotic effects of NF-κB is compared to the cDNA library of acontrol cell that was not exposed to the anticancer treatment. cDNAclones representing genes that are induced by activated NF-κB are thenidentified. The cDNA clones may then be used to isolate thecorresponding genomic clone.

Genes induced by NF-κB that suppress apoptosis (i.e, have anti-apoptoticeffects) can be used in screening assays to identify compounds thatinhibit the anti-apoptotic effects of the genes. Such a method wouldcomprise comparing cells exposed to activated NF-κB and test compound,and cells that were exposed to activated NF-κB only. Compounds thatreduce or prevent the anti-apoptotic effects of the identified gene maybe useful in enhancing the cytotoxic effects of anticancer therapies, orbe useful as anticancer agents themselves.

Thus a further method of the present invention is the co-administrationof an anti-cancer treatment with a compound that reduces or prevents theeffects of an NF-κB-induced anti-apoptotic gene. Such a compound may actto prevent transcription or translation of the anti-apoptotic gene, orinteract with the gene product.

The present invention also provides pharmaceutical compositions for usein the treatment of cancer and neoplastic diseases. The preferredembodiment compositions comprise mixtures of an antineoplastic agent anda suitable NF-κB inhibitor, with suitable pharmaceutical carriers anddiluents. Thus, a preferred embodiment composition comprises: (a) aneffective amount of an NF-κB inhibitor and (b) an effective amount of achemotherapeutic agent.

Pharmaceutical compositions in accordance with the present invention maybe administered by any suitable means, as would be apparent to one ofordinary skill in the art, including orally (in the form of tablets,capsules or solutions), or parenterally (in the form of injections orpellets). Such preparations can be made using known pharmaceuticallyaccepted carriers and excipients. For administration, the compositionsof the present invention will generally be mixed, prior toadministration, with a non-toxic pharmaceutically acceptable carriersubstance (e.g., normal saline or phosphate-buffered saline), and may beadministered using any medically appropriate procedure including oral,intravenous, intraarterial, intradermal, intracavity and intrathecaladministration, and direct injection into the tissue to be treated.

Subjects suitable for treatment according to the present invention arepreferably mammals, and include humans, non-human primates, andnon-primate mammals (including veterinary and livestock subjects), thatsuffer from a neoplastic disease or tumor, including but not limited tolung, breast, prostate, brain, kidney, liver, spleen, pancreas, bone andmuscle cancers. Subjects may have solid or cystic tumors, or diffusedisease.

The methods disclosed herein may be used in subjects having solid orcystic tumors, such as brain tumors or tumors residing in other solidorgans. Treatment may comprise administration of NF-κB inhibitors to thetissue of a solid tumor or to the cystic cavity of a cystic tumor (thatis, a tumor surrounding a fluid-filled cavity), or may comprise surgicalremoval of the solid tumor with administration of NF-κB inhibitor to theresection cavity created by the surgery. Administration of NF-κBinhibitors according to the present invention is in conjunction withchemotherapy or radiation therapy suitable for treatment of the targetneoplasm.

Any type of cancer, tumor or neoplasia (both benign and malignant) maybe treated by the methods of the present invention, including sarcomas,carcinomas, and mixed tumors. Exemplary conditions that may be treatedaccording to the present invention include fibrosarcomas, liposarcomas,chondrosarcomas, osteogenic sarcomas, angiosarcomas, lymphangiosarcomas,synoviomas, mesotheliomas, meningiomas, leukemias, lymphomas,leiomyosarcomas, rhabdomyosarcomas, squamous cell carcinomas, basal cellcarcinomas, adenocarcinomas, papillary carcinomas, cystadenocarcinomas,bronchogenic carcinomas, melanomas, renal cell carcinomas,hepatocellular carcinomas, transitional cell carcinomas,choriocarcinomas, seminomas, embryonal carcinomas, wilm's tumors,pleomorphic adenomas, liver cell papillomas, renal tubular adenomas,cystadenomas, papillomas, adenomas, leiomyomas, rhabdomyomas,hemangiomas, lymphangiomas, osteomas, chondromas, lipomas and fibromas.

According to the present invention, NF-κB inhibitors are utilized asadjuncts in the chemotherapy or radiotherapy of neoplastic disease. TheNF-κB inhibitor is provided in a therapeutically effective amount (i.e.,an amount sufficient to increase or enhance the cytotoxic effectivenessof the co-administered chemotherapeutic agent or radiation treatment),compared to that which would occur in the absence of NF-κB inhibitor.The use of NFκB inhibitors according to the present method enhances thecytotoxic effects of cancer therapies by preventing activation of NF-κB,thus preventing the protective (anti-apoptotic) effects of NF-κB.Cytotoxicity may be assessed by a reduction in the proliferation ofcells and/or decreased numbers of viable cells, leading to a totaldecrease in the number of viable cells.

As used herein, a therapeutically effective amount of an NF-κB inhibitorrefers to those dosages or amounts which decrease or prevent theactivation of NF-κB such that the treated cell, or treated population ofcells, is more susceptible to the apoptotic effects of a cytotoxicchemotherapy or radiation therapy. Increased susceptibility of cells maybe evidenced by decreased cell survival compared to control cellstreated without the co-administration of an NF-κB inhibitor; or by theability to decrease the dosage of the co-administered chemotherapeuticagent or radiation treatment, compared to the dosage required in controlcells treated without co-administered NF-κB. Effective dosages of NF-κBinhibitors according to the present invention may vary depending uponthe specific NF-κB inhibitor used, the time of administration, thecondition being treated, the route of administration, theco-administered cytotoxic agent, and the general condition of thesubject being treated. In vitro and animal models to determineparticular effective dosages are available and apparent to those ofskill in the art. The use of NF-κB inhibiting agents to enhance thecytotoxicity of agents used in the treatment of neoplastic cell growthwill be a useful adjunct in treating tumors, cancers, and otherneoplastic conditions.

Suitable dosages of NF-κB inhibitors will vary as discussed above, butmay range from about 0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg or 0.3 mg/kg; toabout 0.3 mg/kg, 0.5 mg/kg, 1.0 mg/kg, or 10.0 mg/kg, or more.

As used herein, the term chemotherapeutic agent refers to cytotoxicantineoplastic agents, that is, chemical agents which preferentiallykill neoplastic cells or disrupt the cell cycle of rapidly proliferatingcells, used therapeutically to prevent or reduce the growth ofneoplastic cells. Chemotherapeutic agents are also known asantineoplastic drugs or cytotoxic agents, and are well known in the art.As used herein, chemotherapy includes treatment with a singlechemotherapeutic agent or with a combination of agents. In a subject inneed of treatment, chemotherapy may be combined with surgical treatmentor radiation therapy, or with other antineoplastic treatment modalities.

Exemplary chemotherapeutic agents are vinca alkaloids,epipodophyllotoxins, anthracycline antibiotics, actinomycin D,plicamycin, puromycin, gramicidin D, paclitaxel (TAXOL®, Bristol MyersSquibb), colchicine, cytochalasin B, emetine, maytansine, and amsacrine(or “mAMSA”). The vinca alkaloid class is described in Goodman andGilman's The Pharmacological Basis of Therapeutics, 1277-1280 (7th ed.1985) (hereafter “Goodman and Gilman”). Exemplary of vinca alkaloids arevincristine, vinblastine, and vindesine. The epipodophyllotoxin class isdescribed in Goodman and Gilman, supra at 1280-1281. Exemplary ofepipodophyllotoxins are etoposide, etoposide orthoquinone, andteniposide. The anthracycline antibiotic class is described in Goodmanand Gilman, supra at 1283-1285. Exemplary of anthracycline antibioticsare daunorubicin, doxorubicin, mitoxantraone, and bisanthrene.Actinomycin D, also called Dactinomycin, is described in Goodman andGilman, supra at 1281-1283. Plicamycin, also called mithramycin, isdescribed in Goodman and Gilman, supra at 1287-1288. Additionalchemotherapeutic agents include cisplatin (PLATINOL® Bristol MyersSquibb); carboplatin (PARAPLATIN®, Bristol Myers Squibb); mitomycin(MUTAMYCIN®, Bristol Myers Squibb); altretamine (HEXALEN®, U.S.Bioscience, Inc.); cyclophosphamide (CYTOXAN®, Bristol Myers Squibb);lomustine [CCNU] (CEENU®, Bristol Myers Squibb); carmustine [BCNU](BICNU®, Bristol Myers Squibb); irinotecan (CPT-11).

Methods of administering chemotherapeutic drugs vary depending upon thespecific agent used, as would be mown to one skilled in the art.Depending upon the agent used, chemotherapeutic agents may beadministered, for example, by injection (intravenously, intramuscularly,intraperitoneally, subcutaneously, intratumor, intrapleural) or orally.

As used herein, the administration of a compound “in conjunction with” asecond compound means that the two compounds are administered closelyenough in time that the presence of one alters the biological effects ofthe other. The two compounds may be administered simultaneously(concurrently) or sequentially. Simultaneous administration may becarried out by mixing the compounds prior to administration, or byadministering the compounds at the same point in time but at differentanatomic sites or using different routes of administration.

The phrases “concurrent administration”, “simultaneous administration”or “administered simultaneously” as used herein, means that thecompounds are administered at the same point in time or immediatelyfollowing one another. In the latter case, the two compounds areadministered at times sufficiently close that the results observed areessentially indistinguishable from those achieved when the compounds areadministered at the same point in time.

Many chemotherapeutic agents act at specific phases of the cell cycle,and are active only against cells in the process of division. Neoplasmswhich are the most susceptible to chemotherapy are those with a highpercentage of cells in the process of division, including but notlimited to breast, liver, brain, lung, and ovarian cancer.

Anti-cancer radiotherapy may be carried out in any conventional manneras is known in the art, including external beam radiotherapy andimplanted radioactive sources (such as temporarily implanted radioactiveiodine sources to deliver high dose local therapy).

Applicants specifically intend that the disclosures of all U.S. patentscited herein be incorporated by reference in their entirety

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

Example 1 Expression of Super-Repressor I_(κ)B_(α) in HT1080I CellsBlocked TNF-Stimulated NF-_(κ)B Nuclear Translocation

HT1080 fibrosarcoma cells were co-transfected with the pCMV empty vectorto produce control cells HT1080V; HT1080, cells co-transfected with thepCMV vector containing a cDNA encoding the super-repressor IκBα and withthe pCEP4 vector for hygromycin B selection (400 μg/ml) provided HT1080Icells that expressed the super-repressor IκBα. Transfection was by thelipofectamine protocol (Gibco/BRL. IκBα levels were determined byimmunoblotting (ECL, Amersham) to equivalent amounts of protein from thedifferent cell lines with an antibody to human IκBα (Rockland, Inc.,Royertown, Pa.). The expression of super-repressor IκBα in HT1080I cellsefficiently blocked TNF-stimulated NF-κB nuclear translocation asdetermined by electrophoretic mobility-shift assay (EMSA) (data notshown).

Example 2 Expression of the Super-Repressor I_(κ)B_(α) EnhancesTNF-Mediated Apoptosis

HT1080V (open squares) and HT1080I cells (solid diamonds) were treatedwith TNFα (20 ng/ml) for varying times and surviving cells werequantified by crystal violet assay Mehlen et al., EMBO J. 15:2695(1996)). Data shown (FIG. 1A) are the mean of three independentexperiments±SD, and the percentage cell survival was defined as therelative number of TNF treated versus untreated cells. Cell survival wasreduced in HT1080I cells compared to HT1080V controls.

TNF-induced apoptosis was detected by TUNEL staining (Graeber et al.,Nature 379:88 (1996)). HT1080V (V) and HT1080I (I) cells were untreated(−TNF) or stimulated (+TNF) with 50 ng/ml TNFα for 7 hr and fixed with4% paraformaldehyde. The staining was done according to themanufacturer's instructions (Boehringer Mannheim). HT1080I cells treatedwith TNF showed the condensed morphology typical of apoptotic cells(FIG. 1B). In the absence of TNF, neither control cells norsuper-repressor IκBα expressing cells showed apoptotic morphology. Inthe presence of TNF, only super-repressor IκBα expressing cells showedapoptotic morphology, indicating that the presence of IκBα enhancedTNFα-induced apoptosis.

Example 3 NF-_(κ)B Suppresses TNF-Induced Apoptosis

To exclude the possibility that the expression of the super-repressorform of IκBα leads to a function that is different from the inhibitionof NF-κB, the requirement for NF-κB in the inhibition of TNF-inducedapoptosis was confirmed. Cells were pre-treated with interleukin-1(IL-1), an activator of NF-κB that does not initiate apoptosis, prior tocombined cycloheximide (CHX) and TNF treatment.

HT1080V and HT1080I cells were preincubated with 10 ng/ml IL-1β (R & DSystems) for 5 hours (+Pre IL-1), or were left untreated. Cells werethen treated with 10 μg/ml cycloheximide (CHX) and differentconcentrations of TNFα (0-50 ng/ml). Surviving cells were quantified bycrystal violet assay (Mehlen et al., EMBO J. 15:2695 (1996)). As shownin FIG. 2A, Interleukin-1 (IL-1) pre-treatment inhibitedTNFα+CHX-induced apoptosis in HT1080V cells. Open squares representHT1080V cells without IL-1 pretreatment; closed squares representHT1080V cells pretreated with IL-1; circles represent HT1080I cellswithout IL-1 pretreatment; triangles represent HT1080I cells pretreatedwith IL-1.

Example 4 Proteasome Inhibitors Enhanced TNF Apoptosis

The effect of a proteasome inhibitor on TNF-treated cells wasinvestigated. Proteasome inhibitors of the peptide aldehyde category arepotent inhibitors of NF-κB activation (Palombello et al., Cell 78:773(1994); Chen et al., Genes Dev. 9:1586(1995)). It was demonstrated that,in a dose-dependent fashion, the proteasome inhibitor MG132(Z-leu-leu-leu-H) strongly enhanced TNF-induced killing of HT1080V cells(data not shown). Cell viability experiments were used to monitor theeffectiveness of MG132 in enhancing TNF-induced cell killing.

Example 5 Restoration of Resistance to TNF Apoptosis by NF-_(κ)B

Expression of the p50 and RelA/p65 NF-κB subunits restored cellresistance to TNP killing in cells expressing the super-repressor IκB.HT1080I cells (expressing the super-repressor IκBα) were eitherco-transfected with pcDNA3-LacZ (Invitrogen) and pCMV-p65 (2 μg) andpCMV-p50 (2 μg) (hatched bars); or with the LacZ and empty vectors.Transfection was by the lipofectamine protocol, After 40 hours, cellswere treated with different concentrations of TNFα for an additional 24hours. The results are from the mean±SD of two experiments.

As shown in FIG. 2B, cell survival after exposure to TNFα was increasedin cells expressing the NF-κB subunits, compared to control cells.

Example 6 Ionizing Radiation and Daunorubicin Induce NuclearTranslocation of NF-κB in Control Cells

Daunorubicin and ionizing radiation induce nuclear translocation ofNF-κB. HT1080V (V) and HT1080I (I) cells were treated with 1 μMdaunorubicin (Sigma), 50 nM staurosporine (Sigma), or were irradiated (5Gy) for the indicated times. EMSA was performed as previously described(Finco et al., Proc. Natl. Acad. Sci. USA, 91:11884 (1994) to assessnuclear translocation of NF-κB. Results of EMSA are provided in FIG. 3A,where lanes 1-5=daunorubicin treatment; lanes 6-9=staurosporinetreatment; and lanes 10-13, ionizing radiation treatment.

As shown in FIG. 3A, after six hours, only slight nuclear translocationof NF-κB was evident in control (HT1080V) cells treated withstaurosporine (lanes 6-9). In control cells treated with daunorubicin(lanes 1-3), translocation was clearly evident at five hourspost-treatment; no translocation was evident in super-repressor IκBαexpressing cells treated with daunorubicin (lanes 4-5). In control cellstreated with ionizing radiation (lanes 10-11), translocation was clearlyevident at five hours post-treatment; no translocation was evident insuper-repressor IκBα expressing cells treated with ionizing radiation(lanes 12-13).

Example 7 Overexpression of I_(κ)B_(α) Enhanced Cell-Killing by IonizingRadiation and Daunorubicin

As indicated in FIG. 3B, HT1080V cells and HT1080I cells (hatched bars)were treated with the varying concentrations of daunorubicin (0.1 to 2.0μM) for 24 hours. Surviving cells were quantified by crystal violetassay (Mehlen et al., EMBO J. 15:2695 (1996). Data shown represent themean of four separate experiments. Cell survival over time was reducedin HT1080I cells in response to daunorubicin, compared to control cells.

Five hundred HT1080V cells and 500 HT1080I cells were plated in separatesix-well plates. Twenty-four hours later the cells were exposed toionizing radiation in doses from 0 Gy to 10 Gy, and fourteen days latercell clones were counted. Each group was performed in triplicate.Results are shown in FIG. 3C and represent three independent experimentsexpressed as the mean±SD. At radiation doses of 5 Gy and 10 Gy, cellsurvival was reduced in HT1080I cells compared to control HT1080V cells.

The above results indicate that the overexpression of super-repressorIκBα in HT1080I cells enhanced cell killing by daunorubicin and ionizingradiation.

Example 8 Augmentation of Cytotoxicity of Vincristine by NF-κBInhibition

The present inventors' preliminary data additionally indicate that cellkilling by vincristine is augmented by the inhibition of NF-κB (data notshown). HT1080V and HT1080I cells were treated with; vincristine andcell viability was determined. Cell killing in HT1080I cells wasenhanced compared to that in HT1080V cells.

Example 9 Adenoviral Delivery of the Super-Repressor I_(κ)B EnhancesCPT-11 Efficacy

The antineoplastic agent CPT-11 (irinotecan) was determined to activateNF-κB (data not shown). The co-administration of the super-repressor IκBwith CPT-11 was investigated in nude mice with experimentally inducedfibrosarcomas. Delivery of the super-repressor IκB was achieved byinjecting tumors directly with an adenovirus encoding thesuper-repressor IκB under the control of the cytomegalovirus (CMV)promoter. A control adenoviral vector contained the CMV promoter but notthe coding sequence for IκB. Animals were systemically treated withCPT-11, according to protocols known in the art.

Treatments were as indicated in FIG. 4, where solid triangles indicateIκB and PBS treatment; solid squares indicate treatment with the controlviral vector (CMV) and CPT-11; closed circles indicate treatment withPBS only; open triangles indicate treatment with the control viralvector and PBS; crosses (X's) indicate treatment with IκB and CPT-11;and asterisks indicate treatment with PBS and CPT-11. (The data pointsfor IκB and PBS treatment (solid triangles), CMV and CPT-11 treatment(solid squares), and PBS treatment (closed circles) were so similar thatthey are indicated as a single line on FIG. 4.)

As shown in FIG. 4, growth of fibrosarcoma tumors in mice was reduced byco-administration of super-repressor IκB and CPT-11 (bottom line ongraph, indicated by X's), compared to tumor growth in animals treatedwith CPT-11 alone (top line, indicated by asterisks). Tumors receivingboth IκB and CPT-11 showed essentially no growth during the 20 daysafter treatment. CPT-induced cell death was by apoptosis (data notshown).

The above results indicate that the efficacy of anti-neoplastictherapies can be enhanced by the concomitant suppression of NF-κBactivation.

1. A method of enhancing the cytotoxic effects of an antineoplasticchemotherapeutic agent, comprising administering to a mammalian subjectin need of said antineoplastic chemotherapeutic agent a therapeuticallyeffective amount of Nuclear Factor-kappa B (NF-κB) inhibitor inconjunction with the administration of a dosage of the chemotherapeuticagent, whereby the cytotoxic effect of said antineoplasticchemotherapeutic agent is increased compared to that which would occurin the absence of NF-κB inhibitor; wherein the dosage of saidantineoplastic chemotherapeutic agent is decreased as compared to adosage effective without administering the NF-κB inhibitor inconjunction with the administration of the chemotherapeutic agent, andwherein said subject is afflicted with lung, breast, prostate, brain,kidney, liver, spleen, pancreas, bone or muscle cancer, leukemia orlymphoma, a sarcoma, a carcinoma or a mixed tumor.
 2. The methodaccording to claim 1 wherein said NF-κB inhibitor is administeredsimultaneously with said chemotherapeutic agent.
 3. The method of claim1 where said chemotherapeutic agent is selected from the groupconsisting of vinca alkaloids, epipodophyllotoxins, anthracycleneantibiotics, actinomycin D, plicamycin, puromycin, gramicidin D,paclitaxel, colchicine, cytochalasin B, emetine, maytansine, amsacrine,cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide,lomustine, carmustine, and irinotecan.
 4. The method according to claim1, wherein said subject is afflicted with lung, breast, prostate, brain,kidney, liver, spleen, pancreas, bone or muscle cancer.
 5. The methodaccording to claim 1, wherein said subject is afflicted with leukemia orlymphoma.
 6. The method according to claim 1, wherein said subject isafflicted with a sarcoma, a carcinoma or a mixed tumor.
 7. The methodaccording to claim 1, wherein said NF-κB inhibitor and saidchemotherapeutic agent are administered sequentially.
 8. The method ofenhancing the cytotoxic effects of an antineoplastic chemotherapeuticagent, comprising administering to a mammalian subject in need of saidantineoplastic chemotherapeutic agent a therapeutically effective amountof Nuclear Factor-kappa B (NF-κB) inhibitor in conjunction with theadministration of a dosage of the chemotherapeutic agent, whereby thecytotoxic effect of said antineoplastic chemotherapeutic agent isincreased compared to that which would occur in the absence of NF-κBinhibitor; wherein the dosage of said antineoplastic chemotherapeuticagent is decreased as compared to a dosage effective withoutadministering the NF-κB inhibitor in conjunction with the administrationof the chemotherapeutic agent, wherein said NF-κB inhibitor is selectedfrom the group consisting of: a proteasome inhibitor, an inhibitor ofubiquitin conjugation, an inhibitor of proteasome peptidases, and aprotease inhibitor.
 9. The method according to claim 8 wherein saidNF-κB inhibitor is administered simultaneously with saidchemotherapeutic agent.
 10. The method of claim 8 where saidchemotherapeutic agent is selected from the group consisting of vincaalkaloids, epipodophyllotoxins, anthracyclene antibiotics, actinomycinD, plicamycin, puromycin, gramicidin D, paclitaxel, colchicine,cytochalasin B, emetine, maytansine, amsacrine, cisplatin, carboplatin,mitomycin, altretamine, cyclophosphamide, lomustine, carmustine, andirinotecan.
 11. The method according to claim 8, wherein said subject isafflicted with lung, breast, prostate, brain, kidney, liver, spleen,pancreas, bone or muscle cancer.
 12. The method according to claim 8,wherein said subject is afflicted with leukemia or lymphoma.
 13. Themethod according to claim 8, wherein said subject is afflicted with asarcoma, a carcinoma or a mixed tumor.
 14. The method according to claim8, wherein said NF-κB inhibitor and said chemotherapeutic agent areadministered sequentially.
 15. The method according to claim 1, whereinsaid subject is afflicted with breast cancer.
 16. The method accordingto claim 15, wherein said chemotherapeutic agent is selected from thegroup consisting of cisplatin, carboplatin, irinotecan, paclitaxel,vinca alkaloids, and cyclophosphamide.
 17. The method according to claim1, wherein said subject is afflicted with lung cancer.
 18. The methodaccording to claim 17, wherein said chemotherapeutic agent is selectedfrom the group consisting of cisplatin, carboplatin, irinotecan,paclitaxel, vinca alkaloids, and cyclophosphamide.
 19. The methodaccording to claim 1, wherein said subject is afflicted with prostatecancer.
 20. The method according to claim 19, wherein saidchemotherapeutic agent is selected from the group consisting ofcisplatin, carboplatin, irinotecan, paclitaxel, vinca alkaloids, andcyclophosphamide.
 21. The method according to claim 1, wherein saidsubject is afflicted with brain cancer.
 22. The method according toclaim 21, wherein said chemotherapeutic agent is selected from the groupconsisting of cisplatin, carboplatin, irinotecan, paclitaxel, vincaalkaloids, and cyclophosphamide.